Bacterial polysaccharide

ABSTRACT

A high-viscosity heteropolysaccharide composed of 33% mannose, 29% glucose, 21% galactose and 17% glucuronic acid, and containing 5.7% acetyl and 4.9% pyruvate.

RELATED APPLICATION

This is a division of application Ser. No. 889,163 filed Mar. 23, 1978,which is a continuation-in-part of application Ser. No. 842,646, filedOct. 17, 1977, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention pertains to a novel heteropolysaccharide which isproduced by the action of a bacteria on a selected carbon source.Further, the invention pertains to a novel process for producing aheteropolysaccharide by bacterial fermentation of a selected carbonsource under controlled conditions.

2. Description of the Prior Art

It is known that heteropolysaccharides can be produced by certainmicroorganisms. Some of such heteropolysaccharides function ashydrophilic colloids and because of their viscosity properties andrheology have been used as thickening agents for aqueous systems.Illustrative of prior art heteropolysaccharides, their preparation anduses are U.S. Pat. Nos. 3,020,207; 3,256,271; 3,894,976; 3,915,800 and3,894,976.

As with other fields of technology, research has continued with theobjective of discovering new heteropolysaccharides having usefulproperties as thickening, suspending and/or stabilizing agents.

OBJECTS OF THE INVENTION

It is an object of this invention to provide a new heteropolysaccharide.It is another object to provide a method for making this new compound. Astill further object is provision of formulations containing our newheteropolysaccharide as a thickening or suspending or stabilizing agent.These and other objects of the invention will be apparent from thefollowing description of this invention.

SUMMARY OF THE INVENTION

It has now been found that a high viscosity anionic heteropolysaccharidecomposed of about 33% mannose, 29% glucose, 21% galactose and about 17%glucuronic acid and also containing about 5.7% acetyl and about 4.9%pyruvate is obtained by an aerobic fermentation of an organism isolatedfrom a soil sample from the Canal Zone. This heteropolysaccharide hasdesirable thickening, suspending and/or stabilizing properties inaqueous systems.

DETAILED DESCRIPTION

The heteropolysaccharide of this invention is a high molecular weightpolysaccharide containing primarily carbohydrate residues and a minoramount of protein. It is sometimes referred to as a "gum" but it isbelieved that the heteropolysaccharide terminology is more accurate andprecise. In the following description of our invention it will sometimesbe referred to as Heteropolysaccharide S-21.

The bacterium employed in the process of the present invention which isidentified as Strain tTR-45, is a mutant of Klebsiella pneumoniae StrainS-21 that was isolated from the rhizosphere soil of a plant of the genusAechmea epiphytes belonging to the pineapple family. The soil sample wasobtained in the Canal Zone. Strain tTR-45 requires thymine for growth at37° C. but does not require thymine for growth at 30° C. A deposit ofStrain tTR-45 was made with the American Type Culture Collection, 12301Parklawn Drive, Rockville, Maryland 20852, on 11 August 1977 underAccession No. ATCC 31314. The culture is available to the public withoutrestriction.

This organism requires a fermentation medium that supplies a carbonsource, a phosphorus source, a nitrogen source, a magnesium source andan iron source. The carbon source typically is hydrolyzed starch with aDE range of 12-31. The starch can be hydrolyzed with commerciallyavailable α-amylases. The phosphorus source may be either Na₂ HPO₄, NaH₂PO₄, K₂ HPO₄ or KH₂ PO₄ or a mixture thereof. The concentration mayrange from about 0.025 to about 0.5%. The magnesium source may besupplied with MgCl₂ or MgSO₄ in concentrations of from about 0.005 toabout 0.02%. The nitrogen source may be NaNO₃, KNO₃, NH₄ NO₃ (NH₄)₂ SO₄,or NH₄ Cl as well as organic sources such as soy peptone Type T(Sheffield Chemical, Norwich, New York), Promosoy 100 (Central SoyaChemurgy Division), NZ-amine Type A (Sheffield), or Ferm Amine Type IV(Sheffield). The medium may contain either inorganic or organic nitrogenor mixtures thereof. The concentration of inorganic nitrogen in themedium may range from about 0.045 to about 0.2% and with the organicnitrogen from about 0.01 to about 0.1%. The iron may be supplied to thefermentation as FeCl₃ or FeSO₄ at levels of 1-10 ppm.

The pH of this fermentation preferably is maintained between about 6.3and about 7.7, and the temperature between about 28° C. and about 33° C.for maximum polysaccharide production.

The fermentation time is typically from about 48 to 60 hours when properconditions of medium, temperature, pH and other fermentation parametersare met.

DETAILS OF PROCEDURES

The soil sample when received is plated onto yeast-malt (YM) agar, E-1agar with 1% dextrose and E-1 agar with 1% 42 dextrose equivalent (DE)corn syrup, and an isolate, Strain 21, is picked from a YM agar plateand pure cultured on nutrient agar.

Strain S-21 is plated on minimal medium containing thymine andtrimethoprim and incubated at 37° C. for 4 days. Those colonies whichgrow on the plate are tested for a thymine requirement at 37° C. Thosestrains that required thymine at 37° C. are tested again for thyminerequirement at 30° C. and 37° C. Strain tTR-45 is one of five strainsthat did not require thymine at 30° C. but did at 37° C. The reversionfrequency on minimal medium is about 2×10⁻⁸ revertants/cell.

This mutant can be cultured on blood agar, chocolate agar, brain heartinfusion agar, and nutrient agar. These rich media all have sufficientthymine to meet the thymine requirement of tTR-45. This mutant does notgrow on minimal medium at 37° C. but will grow on minimal medium withadded thymine. The minimum thymine requirement is greater than 5 ppm butless than 10 ppm.

The minimal medium is prepared as follows:

    ______________________________________                                        Salt Solution                                                                 ______________________________________                                        K.sub.2 HPO.sub.4   10.5 gm.                                                  KH.sub.2 PO.sub.4   4.5 gm.                                                   (NH.sub.4).sub.2 SO.sub.4                                                                         1.0 gm.                                                   Sodium citrate . 2H.sub.2 O.                                                                      0.5 gm.                                                   Water               470 ml.                                                   ______________________________________                                    

Autoclave the above medium. Then add 1 ml. of a sterile 1 M solution ofMgSO₄.7H₂ O, 10 ml. of a 20% sterile glucose solution and when needed,10 ml. of a sterile 0.5% solution of thymine and 10 ml. of a 0.5%solution of trimethoprim. Add solution to 15 gm. sterile agar in 500 ml.H₂ O, mix and pour plates.

Urine agar was prepared by diluting sterile urine with an equal volumeof agar solution. This did not support the growth of tTR-45 at 37° C.

Tissue medium was prepared in the following manner. Rabbit kidney, lungand liver were removed quickly from a sacrificed animal, cooled in ice,homogenized in an equal volume of water, and autoclaved. Afterautoclaving, the tissue homogenate was mixed with an equal volume ofagar solution and plates poured from this. The total dilution of tissuewas 1:4. These media did not support the growth of tTR-45 at 37° C. butdid at 30° C. This same media prepared from bovine kidney also did notsupport the growth of tTR-45 at 37° C. but did at 30° C.

In another experiment in which the liver and lung were not cooledrapidly, and the homogenate allowed to stand at room temperature forabout 15 minutes before autoclaving, the media supported the growth oftTR-45 at 37° C. This could have resulted from the liberation ofthymidine from the DNA of the organ homogenates by DNases found in thetissues.

Shake flask experiments are done in 500 ml. unbaffled Erlenmeyer flasks.The flasks are incubated on a New Brunswick Model V gyrotary shakeruntil harvesting. The fermentation temperature is 30° (all temperaturesthroughout this specification and claims are expressed in degreesCelsius unless indicated otherwise). The product identified asheteropolysaccharide S-21, or more simply S-21, is recovered byprecipitation with 2-3 volumes of isopropyl alcohol (IPA), the fiberscollected and dried at 55°-65° overnight. One percent reconstitutedviscosities are obtained by dissolving two grams of product in 198 gramsof deionized water using a Lightnin' mixer. Unless otherwise stated, allviscosities are measured using a Brookfield Model LVF viscometer withthe No. 4 spindle at 60 rpm. S-21 gum contains from about 30 to about37% mannose, from about 26% to about 32% glucose, from about 19% toabout 23% galactose and from about 15.3% to about 18.8% glucuronic acid.It has an acetyl content of from about 5.1% to about 6.3%, and apyruvate content of from about 4.5% to about 5.4%.

The 20 L fermentor scale-ups are done is Fermentation Design fermentors.The fermentations are started at an air rate of 10 L/M and an agitationrate of 200-300 rpm. At 16-24 hours the agitation is increased to 800rpm. The pH is controlled at 6.6-6.7 with 30% KOH. The fermentationtemperature is 30°. The fermentation liquor is pasteurized at 80° for 10minutes before harvesting.

The taxonomic data on dextrose, lactose, lysine decarboxylase, ornithinedecarboxylase, urea and citrate (Simmon's) is obtained using theEnterotube (Roche Diagnostics) system. The malonate, nitrate reduction,esculin hydrolysis, phenylalanine deaminase and Voges-Proskauer are doneusing the Pathotec (General Diagnostics) test system. Phenylalaninedeaminase is also checked in the standard medium for this test accordingto the method of Harrigan, W. F. and M. E. McCance, Laboratory Methodsin Microbiology, Academic Press, 1966, p. 296. Hydrogen sulfideproduction is tested in Triple Sugar Iron (TSI) agar and also incysteine broth with lead acetate paper strips, ibid., p. 55. Thefermentation of sucrose, fructose, cellobiose, inositol and dulcitol wschecked in Dye's low buffer carbohydrate fermentation medium. Motilityis checked on cells from 20-24 hours NA slants by the hanging dropmethod and by flagellar stains according to the method of Rosen, A. andR. E. Levin, Vibrios from Fish Pen Slime Which Mimic Escherichia coli onViolet Red Bile Agar, Appl. Microbiol., 20:107-112, 1970. Motility isalso checked in semi-solid agar.

Alkaline stability is checked by dissolving 2 grams of gum in 160 ml oftap water. Then while mixing, 40 ml of 50% NaOH is added. The viscosityis measured and the samples stored at 43° for seven days. Acid stabilityis checked by dissolving 4 grams of gum in 147 ml of tap water. Then 53ml of concentrated HCl are added slowly with good mixing. The viscosityof the samples are measured and the samples stored at 43°. Thermalstability is measured by autoclaving a 1% solution of gum at 15 psi (2atomospheres) and 121° for 15 minutes followed by rapid cooling.Viscosities are measured before and after heating.

Temperature-viscosity curves are obtained by heating the gum solution inan oil bath or cooling it in an ice bath and then removing the samplefrom the respective bath and measuring the viscosity. Concentration vs.viscosity curves are obtained by making individual solutions of gum indeionized water and measuring their viscosity.

The effect of pH on the viscosity of a 1% solution is checked byadjusting the pH with HCl or KOH, allowing the sample to set for 15minutes, then measuring the viscosity. The effect of added electrolyteis checked by adding various concentrations of NaCl to a 1% solution andmeasuring the viscosity.

Solvent solubilities are obtained by trying to dissolve the gum invarious solvents.

The methylene blue compatibility test is carried out by adding 100 ml ofa 1% solution of gum to a mixture of one gram of methylene blue in 3.0ml of glacial acetic acid. The gum is incompatible if it precipitates.

RESULTS: A. Taxonomy

The taxonomic results are shown in Table 1. The organism is a gramnegative, non-motile, non-spore forming rod. It produces acid and gasfrom glucose, lactose, sucrose, cellobiose and fructose. It producesacid from inositol but does not ferment dulcitol. The organism ispositive for the Voges-Proskauer test, lysine decarboxylase, urease, andnitrate reductase. The organism produces enough H₂ S for a positive testwith the lead acetate test procedure, but is negative for H₂ S on TSIagar which is less sensitive. This organism can use citrate or malonateas the sole carbon source. It does not produce ornithine decarboxylaseor phenylalanine deaminase and does not hydrolyze esculin.

On YM agar with organism produces circular, entire, convex colonieswhich are very mucoid. On nutrient agar the colonies are similar butsmaller and less mucoid. No pigment production is observed.

                  TABLE 1                                                         ______________________________________                                        TAXONOMIC CHARACTERISTICS OF STRAIN tTR-45                                    Characteristic        Results                                                 ______________________________________                                        Gram Stain            Gram (-) rod                                            Size                  1 × 2 μ                                        Motility              Non-motile                                              Carbohydrates                                                                 Glucose               Acid and Gas                                            Lactose               Acid and Gas                                            Sucrose               Acid and Gas                                            Cellobiose            Acid and Gas                                            Fructose              Acod and Gas                                            Inositol              Acid Acid                                               Dulcitol              No Change                                               Voges-Proskauer       +                                                       H.sub.2 S Production                                                          Lead Acetate          +                                                       TSI                   -                                                       Lysine Decarboxylase  +                                                       Ornithine Decarboxylase                                                                             -                                                       Phenylalanine deaminase                                                                             -                                                       Urea                  +                                                       Citrate               +                                                       Malonate              +                                                       NO.sub.3.sup.31 Reduction                                                                           +                                                       Esculin Hydrolysis    -                                                       ______________________________________                                    

The fact that Strain tTR-45 is a gram negative, non-spore forming rodcapable of producing acid and gas from lactose indicates it belongs tothe family Enterbacteriaceae. This organism does not produce H₂ S on TSIagar, does not produce phenylalanine deaminase, does produce urease andis positive for the Voges-Proskauer test.

B. Fermentation Improvement

The results in Tables 2 and 3 show the medium optimization work done inshake flasks. The results of the first two experiments indicate that E-1medium is the best, followed by E-1 supplemented with 5 ppm Fe⁺⁺ and inwhich the NH₄ NO₃ is replaced by 0.19% NaNO₃. Calcium carbonate is notan effective substitute for K₂ HPO₄. Doubling the NH₄ NO₃ concentrationhas a very deleterious effect on gum production. However, reduction ofthe NH₄ NO₃ concentration by 50% does not drastically effect gumproduction.

The results with starch hydrolysates indicate that starch with very lowdextrose equivalents (DE) can be used in this fermentation. Theseresults indicate a DE range of 13.4 to 31 can be used for thisfermentation. Corn syrup (42 DE) give a high yield but results in aproduct with a much lower viscosity.

The results of two typical 20 L fermentor scale-ups are shown in Table4. These results indicate that the fermentation should be complete by52±3 hours with 3% carbon source. The conversion efficiency is about60%.

TABLE 2 MEDIUM OPTIMIZATION

Seeds are started in YM broth flasks and incubated for 24 hours at 30°on a gyrotory shaker. One percent inoculums are used to inoculateexperimental flasks containing medium variations. The carbon source ishydrolyzed starch. The starch DE for experiment I is 21.1 and 17.5 forexperiment II. Experiment I is incubated at 30° for 96 hours on agyrotory shaker while experiment II goes for 72 hours.

    __________________________________________________________________________                                 1%                                                                   Beer Gum Reconstituted                                                        Viscosity                                                                          Yield                                                                             Viscosity                                        Medium              (cps)                                                                              (1%)                                                                              (cps)                                            __________________________________________________________________________    I E-1*              100,000+                                                                           1.76                                                                              3100                                               E-1 - NH.sub.4 NO.sub.3 +0.19% NaNO.sub.3                                                       6,800                                                                              1.52                                                                              2450                                               E-1 - K.sub.2 HPO.sub.4 + 0.5% CaCO.sub.3                                                       5,800                                                                              2.04                                                                              --                                                 E-1 - NH.sub.4 NO.sub.3 + 0.19% NaNO.sub.3 +                                  5 ppm Fe.sup.++   80,000                                                                             1.64                                                                              3100                                             II                                                                              E-1               100,000+                                                                           1.98                                                                              3225                                               E-1 + 5 ppm Fe.sup.++                                                                           100,000+                                                                           1.99                                                                              3200                                               E-1 - NH.sub.4 NO.sub.3 + 0.19% NaNO.sub.3 +                                  5 ppm Fe.sup.++   82,500                                                                             1.94                                                                              2650                                               E-1 - NH.sub.4 NO.sub.3 + 0.045% NH.sub.4 NO.sub.3                                              67,000                                                                             1.72                                                                              2600                                               E-1 - NH.sub.4 NO.sub.3 + 0.18% NH.sub.4 NO.sub.3                                                 700                                                                              1.06                                                                              --                                               __________________________________________________________________________     *E-1 medium is composed of 0.5% K.sub.2 HPO.sub.4, 0.09% NH.sub.4             NO.sub.3, 0.05% Promosoy 100, and 0.01% MgSO.sub.4 . 7H.sub.2 O in tap        water.                                                                   

TABLE 3 CARBON SOURCES FOR STRAIN tTR-45

The inoculum is grown in YM broth for 24 hours at 30° on a gyrotoryshaker. A 1% inoculum is used to inoculate flasks containing E-1medium--NH₄ NO₃ +0.19% NaNO₃ +5 ppm Fe⁺⁺ and the following carbonsources. The starch is hydrolyzed with Tenase (Miles). All stachescontain the same enzyme concentration with the DE being varied byaltering heating temperatures and times. The flasks are incubated for 72hours at 30° on a gyrotory shaker.

    ______________________________________                                                      Beer        Gum       %                                                       Viscosity   Yield     RV                                        Carbon Source (cps)       (%)       (cps)                                     ______________________________________                                        Corn Syrup 42 DE                                                                            65,000      2.09      2000                                      Starch DE 13.4                                                                               100,000+   1.81      3600                                      Starch DE 21.7                                                                              96,750      1.88      3250                                      Starch DE 26.7                                                                              100,000     1.87      3150                                      Starch DE 30.9                                                                              81,500      1.73      2850                                      ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        SCALE-UP OF STRAIN tTR-45 TO 20L FERMENTORS                                   EXPERIMENT I     EXPERIMENT II                                                                     Resi-                  Resi-                                  Beer            dual       Beer        dual                                   Vis-    Gum     Carbon     Vis-  Gum   Carbon                            Age  cosity  Yield   Source                                                                              Age  cosity                                                                              Yield Source                            (hrs)                                                                              (cps)   (%)     (%)   (hrs)                                                                              (cps) (%)   (%)                               ______________________________________                                        20   130     --      --    24   1,500 1.53  0.55                              41   6,700   1.96    0.32  48   8,200 1.90  0.15                              65.5 93,500  1.81    Com-  68   18,600                                                                              1.81  Com-                                                   plete                  plete                             1% Product Viscosity -                                                                         1% Product Viscosity -                                       3325 cps         2950 cps                                                     ______________________________________                                         The medium consists of 0.05% K.sub.2 HPO.sub.4, 0.19% NaNO.sub.3, 0.01%       MgSO.sub.4 . 7H.sub.2 O, 0.05% Promosoy 100 and 3.0% starch. The seed         medium is the same with the inoculum rate at 5%. The fermentation             temperature is 30°. Agitation and aeration are increased as            necessary. The pH is controlled at 6.4-6.6 with 30% KOH. The starch DE is     12 in the first experiment and 19 in the second.                         

The results indicate that Strain tTR-45 has a good fermentation(conversion efficiency of 50-67%) in an economic medium with afermentation time of less than 72 hours. The organism will not producegum from dextrose efficiently, as an excess of acid is produced.However, a wide range of starch hydrolysates (DE 12.9-31) can be used asa carbon source. The use of corn syrup (42 DE) results in a lowviscosity product.

The product is easily recovered as fibers by precipitation with 2-3volumes of isopropyl alcohol.

C. Gum Properties

Also, S-21 has little or no viscosity synergism with locust bean gum.S-21 exhibits no acid or alkaline stability (97-100% viscosity loss).Upon autoclaving at 121° and 15 psi (2 atmospheres) for 15 minutes, itloses 39-46% of its viscosity compared to little or no loss for xanthangum.

S-21 is soluble in ethylene glycol but insoluble in isopropyl alcohol,ethyl alcohol, methyl alcohol, acetone, and glycerol. It is alsoincompatible with methylene blue chloride.

The gum is anionic in nature judged by its incompatibility with acationic dye such as methylene blue.

The gum produced by S-21 is an unusually high viscosity gum. It has aviscosity much higher than that of xanthan gum.

Very little viscosity change is noted in a 1% solution of S-21 gum inthe pH range of 4-10. The S-21 gum loses essentially all of itsviscosity in acid or alkaline stability tests. It demonstrates, however,an appreciable degree of thermal stability as less than half of itsoriginal viscosity is destroyed by autoclaving.

The S-21 gum is very slightly sensitive to the addition of NaCl to a 1%gum solution.

The S-21 gum is somewhat temperature sensitive and loses approximately17 cps/°C. in a 1% solution. A 0.5% solution is more temperaturesensitive than a 1.0% solution.

At low concentration levels such as 0.1%, S-21 gum has a much higherviscosity than xanthan gum in deionized water and similar viscosities intap or brine water. At equivalent concentrations of from about 0.4% toabout 1.4%, S-21 has a higher viscosity than xanthan gum.

TABLE 5 VISCOSITIES AT 0.1% CONCENTRATION

The viscosity is measured on a Brookfield Model LVF viscometer using theUL adapter.

    ______________________________________                                                     S-21      Xanthan Gum                                            ______________________________________                                        Deionized H.sub.2 O                                                                          51.5 cps    29   cps                                           Tap H.sub.2 O  21    cps   23   cps                                           Brine H.sub.2 O                                                                               8   cps     5.5 cps                                           ______________________________________                                    

The results in Table 5 show a comparison of 0.1% viscosities indeionized water, tap water and brine water for S-21 and xanthan gum. Theresults are similar except that the deionized water viscosity for S-21is much higher than that of xanthan gum.

TABLE 6 RHEOLOGICAL DATA

                  TABLE 6                                                         ______________________________________                                        RHEOLOGICAL DATA                                                                       S-21     S-10       S-7                                              ______________________________________                                        Thixotropy 1.06       1.08       1.06                                         Yield Point                                                                              27.2       6.6        33                                           Low pseudo-                                                                   phlasticity                                                                              142        80         160                                          High pseudo-                                                                  phlasticity                                                                              388        172        459                                          ______________________________________                                    

The data in Table 6 represent a comparison of rheological data for S-21,S-10 (described in U.S. Pat. No. 3,933,788) and S-7 (described in U.S.Pat. No. 3,960,832). The thixotropy is about the same for all threegums. The yield point for S-21 very close to that of S-7. Thepseudoplasticity values for S-21 are closer to S-7 than to those ofS-10.

D. Gum Component Analysis

Five mg of S-21 gum are added to 2 ml of IN H₂ SO₄ ; the tube is sealedand heated to 100° in a boiling water bath for 8 hours. The tube isopened and the solution neutralized with BaCO₃. The barium sulfate isremoved by filtration and barium ions with H⁺ -charged Amberlite IR 120.The solution is concentrated to a syrup under reduced pressure at 35°. Atentative identification of the sugars present is made by paperchromatography. Alditols are formed by reacting the hydrolysate withsodium borohydride in water overnight. Excess sodium borohydride isremoved by treatment with Amberlite IR-120 (H⁺) and residual boric acidremoved as volatile methyl borate by co-distillation several times withmethanol. Alditol acetates are formed by reacting with acetic anhydridein pyridine overnight. Water is added to the reaction mixture which isthen concentrated to small volume and co-distilled several times withchloroform. The residue is dissolved in chloroform for GLC analysis. GLCis performed with a Hewlett-Packard Model 5750 chromatography using 3%by weight of ECNSS-M on 80/100 Gas Chrom Q at 185°. Sugars areidentified by comparison with authentic standards and the proportions ofalditol acetates are determined directly from the peak areas on the gaschromatograph by integration.

Uronic Acid Content

The uronic acid content is determined by decarboxylation with 19%hydrochloric acid. Liberated carbon dioxide is trapped in standardsodium hydroxide and determined by back-titration.

Acetyl Content

The acetyl content is determined by deacetylating the gum (0.1%solution) in an oxygen-free atmosphere with a known volume of 0.01 Npotassium hydroxide containing 1% (W/V) potassium chloride at roomtemperature. Aliquots are removed at elapsed time intervals and theacetyl content is determined by back-titration with 0.01 N sulfuric acidusing a pH meter.

Pyruvic Acid

The polysaccharide (2 mg/ml) is mixed with an equal volume of 0.2 N-HCland hydrolysed at 100° for 4 hours.

To a cuvette (1 cm path length) containing 2.4 ml of triethanolamine isadded 0.5 ml sample of hydrolysate and 0.1 ml of NADH solution. Aftermixing, the absorbance is read at 340 nm. Approximately 10 μl (2 units)lactate dehydrogenase is added and the absorbance when constant againdetermined.

Calculation

Molar extinction coefficient of NADH=6.22.

Pyruvate content of 0.5 ml sample= ##EQU1##

The results of the foregoing analyses are as follows:

    ______________________________________                                        Mannose             33%                                                       Glucose             29%                                                       Galactose           21%                                                       Glucuronic Acid     17%                                                       Acetyl content      5.7%                                                      Pyruvate content    4.9%                                                      ______________________________________                                    

S-21 gum tends to lump or agglomerate when wet with water. The timerequired to effect complete solution of the biogum depends on the amountof lumping that occurs initially during preparation of the solutions. Ithas been found, however, that the dispersibility of S-21 gum is improvedby heating the gum in the presence of from about 0.05 weight % to about5 weight % of a dialdehyde based on the dry weight of S-21 gum. The gummay be treated with the dialdehyde in dry state or in an aqueous medium.The aqueous medium conveniently may be the fermentation beer in whichthe S-21 gum has been prepared.

The aldehyde may be an aliphatic dialdehyde of from 2 up to about 8carbon atoms, e.g., glyoxal, malonaldehyde, succinaldehyde,glutaraldehyde, adipinaldehyde or octandialdehyde, or polyglyoxal, anoligomeric form of glyoxalhydrates having from 5 to 10 glyoxalhydraterepeating units. Glyoxal is preferred.

The dialdehyde is added to the beer at about the completion of thefermentation process, preferably with agitation to obtain uniformdistribution of the dialdehyde. The beer is then heated to a temperatureof from about 70° C. to about 100° C. for a time sufficient to improvethe dispersibility of the recovered biogum. In general, this takes atleast about 2 minutes, typically from about 2 to about 5 minutes. Whilelonger heating times may be employed, no additional improvement is seenin dispersibility after about 5 minutes of heating.

Heating in the presence of a dialdehyde at a pH below about 7 to obtainimproved dispersibility according to the present invention is mostconveniently and economically carried out by combining the dialdehydetreatment with pasteurization by heating. In this way no additional timeor heating expense is required for the dialdehyde treatment beyond thecost of the dialdehyde itself. While the foregoing description hasdescribed the present invention with reference to a fermentation beer,it will be obvious to those skilled in the art that the invention is notlimited to fermentation beers but that is is applicable to any aqueoussolution of S-21 gum.

The following examples illustrate the present invention without,however, limiting the same thereto.

EXAMPLE 1

Two 500 ml unbaffled shake flasks containing a modified E-1 medium with3% hydrolyzed starch as the carbon source are inoculated with aone-colony inoculum of Strain tTR-45 from 48 hours YM agar plates. Theseflasks are incubated at 30° for 24 hours on a New Brunswick Scientificgyrotory shaker Model V at 300 rpm. They are then used to inoculate a 5L fermentor vessel containing 3 L of the same media. This fermentor hasan aeration rate of 1 L/min. with a tip speed of 263 ft/min. At 24 hoursthis one gallon vessel has a viscosity of 4700 cps and is used toinoculate a 30 L fermentor containing a final volume of 20 L of the samemedium. One liter of seed is used as an inoculum. The dextroseequivalent of the hydrolyzed starch is 12. The aeration rate is 10 L/minwith a tip speed of 234 ft/min. At 20 hours the viscosity of thefermentation liquor is 130 cps and the tip speed is increased to 470ft/min. By 41 hours the viscosity of the fermentation liquor hasincreased to 6700 cps, and the tip speed is increased to 940 ft/min. Thefermentation is complete by 65.5 hours with a viscosity of 93,500 cpsand a yield of 1.81 gms of product per 100 grams of fermentation liquor.The pH of the fermentation is maintained between 6.6 and 7.7 using 30%KOH and an automatic pH control system. The gum yield, determined byweighing out 100 grams of fermentation liquor followed by the additionof 2-3 volumes of isopropanol and vigorous shaking, is 1.81 L g. Theresulting fibers are collected by filtration and dried at 105° overnightprior to weighing. The remaining fermentation liquor is treated in thesame manner except that the product is dried overnight at 55° prior tomilling to a powder. The resulting product has a viscosity of 3325 cpswhen reconstituted at 1% concentration in deionized water.

EXAMPLE 2

A wall joint cement is formed by adding the following blend:

    ______________________________________                                        CaCO.sub.3       383.0 g                                                      Mica P-80-F      75.0                                                         CMC              2.5                                                          Attagel 40       5.0                                                          Bentone LT       2.5                                                          ______________________________________                                    

slowly to a solution of 4 g of S-21 in 275 g of water using a HobartMixer, and then adding 45 g of a latex emulsion.

EXAMPLE 3

A semigloss white latex paint is prepared from the followingformulation:

    ______________________________________                                        Material        Pounds       Gallons                                          ______________________________________                                        Water           72.5         8.7                                              Dowicil 75      2.0          0.17                                             Tamol 731 25%   9.0          7.00                                             Propylene Glycol                                                                              60.0         1.00                                             Ethylene Glycol 22.0         2.25                                             Carbitol Solvent                                                                              18.0         2.25                                             Foamaster G     2.0          0.25                                             Tipure R-900    270.0        8.00                                             Hexylene glycol 10.0         1.31                                             Aerosol OT 75% (aq)                                                                           2.0          0.50                                             Letdown                                                                       Rhoplex AC 490  433.0        49.50                                            Foamaster G     3.0          0.39                                             2.5% aqueous solution                                                         of S-21         178.0        20.75                                            TOTAL           1080.5       102.68                                           ______________________________________                                    

The foregoing paint has a unexpected improvement in flow and levelingproperties as well as gloss compared to a similar paint formulationviscosified with hydroxyethyl cellulose.

EXAMPLE 4

The pH of a fermentation beer sample (31 liters) prepared as describedin Example 1 which contains 1.68 g S-21 per 100 ml of beer is adjustedto 6.3 by addition of concentrated HCl. Glyoxal (40%) is added to aglyoxal level of 3.0% based on weight of gum. After thorough mixing thebeer is heated to 80° C., held for 3 minutes at this temperature andthen cooled to room temperature. The gum in the pasteruized beer isprecipitated by adding 3 volumes of isopropanol, dried in a steam drierat 71° C. and milled. Three grams of the milled gum and 3 grams of acontrol sample from the same beer treated similarly except omitting theglyoxal treatment are sprinkled evenly over the surface of 297 mldistilled water contained in a 400 ml beaker with a stirrer in place.After 30 seconds the stirrer, which rotates at about 800 rpm, isswitched on for one minute. The stirring is then stopped and thesolution is screened through a 20-mesh (U.S. Standard) screen, and thewet material remaining on the screen is weighed. The following resultsare obtained:

    ______________________________________                                        Sample       Weight of Material on Screen (g)                                 ______________________________________                                        Glyoxal treated                                                                            0                                                                Control      22.8                                                             ______________________________________                                    

What is claimed is:
 1. A process for producing Heteropolysaccharide S-21containing from about 30% to about 37% mannose, from about 26% to about32% glucose, from about 19% to about 23% galactose and from about 15.3%to about 18.8% glucuronic acid and having an acetyl content of fromabout 5.1% to about 6.3% and a pyruvate content of from about 4.5% toabout 5.4% that comprises growing the organism ATCC 31314 in an aqueousnutrient medium under submerged aerobic conditions and recovering saidHeteropolysaccharide S-21.
 2. A process according to claim 1 wherein thepH is controlled at from about 6 to about
 7. 3. A process according toclaim 1 wherein the temperature is controlled at from about 25° C. toabout 35° C.
 4. A fermentation beer containing from about 1.5% to about2.1% of heteropolysaccharide S-21.